An attenuated aro A- strain of Salmonella typhimurium, SL3235, was previously shown to afford excellent protection in C3HeB/FeJ mice against challenge by virulent Salmonella and cross-protection against Listeria monocytogenes. In the present study, the immunologic status of immunized mice was evaluated by studying their ability to mount in vivo and in vitro plaque-forming cell responses to SRBC. SL3235-immunized mice exhibited marked suppression in their ability to generate anti-SRBC plaque-forming cell responses. Suppression was active and mediated by soluble factors as demonstrated by the capacity of immune cells to suppress the responses of normal cells in co-culture and across a membrane in transwell plates. Depletion of T cells from immune spleens did not alleviate the suppressive activity of the remaining cells. Depletion of splenic adherent cells resulted in partial alleviation of suppressive activity, demonstrating that mature macrophages are partly responsible for mediating the observed suppression. A sequential multi-step depletion procedure resulted in marked enrichment of a second suppressor cell population that was Mac1+, Thy-1.2-, surface Ig-, J11d-, non-adherent, non-phagocytic, and esterase negative. When cultured in vitro in the presence of L cell-conditioned medium, but not in the presence of Con A supernatant, these cells matured into typical macrophages within 72 h of culture. The cell population enriched for macrophage precursors (75%) retained the suppressive capacity of unfractionated splenocytes. Our data indicate that, in addition to the well-described involvement of mature macrophages in suppressing immune responses, bacterial infection may induce appearance of macrophage precursors that may also play an important regulatory role in the immune system.